Method of screening prion disease infection factor

ABSTRACT

Early detection of infectious agents of human prion diseases such as CJD by using an animal model, etc. is needed in order to rapidly determine prion infections in pharmaceuticals such as blood products, foods, or cosmetics. This invention provides a screening method for infectious agents of human or non-human prion diseases in samples, which employs, as an indication, the deposition of the aberrant prion protein in the follicular dendritic cell (FDC) of a non-human animal.

TECHNICAL FIELD

[0001] The present invention relates to a screening method forinfectious agents of human or non-human prion diseases in samples, novelrecombinant prion proteins, and transgenic animals and knock-in animalsthat express the aforementioned proteins.

BACKGROUND ART

[0002] Creutzfeldt-Jacob Disease (hereinafter abbreviated to “CJD”) isan incurable nervous disease which gives rise to presenile dementia.Once a patient develops the disease, the patient enters a vegetativestate, a so-called bedridden state, in about 3 to 5 months, andeventually dies. At present, there is no effective therapy for CJD, andonly a symptomatic treatment is provided.

[0003] CJD is known not only as a neurodegenerative disease that simplydevelops mental deterioration but also as a disease that is transmittedfrom human to human or from animal to animal. Examples of human to humantransmission are: the kuru disease, which is considered to betransmitted by eating a human brain; CJD transmitted by a growth hormonepreparation; and CJD after dura mater transplantation, which has beenrecently an issue of concern. New variant CJD (nvCJD), which has been anissue of concern in Britain, is considered to arise from thetransmission of bovine spongiform encephalopathy (abbreviated to “BSE”and generally referred to as “mad cow disease”) from bovine to human.

[0004] Based on the assumption that the infectious agent of CJD is madeof protein, the infectious agent was designated as a “prion” in 1982. In1985, the prion protein gene that constitutes the infectious agent,i.e., a prion, was cloned. As a result, it was demonstrated that a prionprotein was also expressed in a normal animal brain. Also, thedeposition of the aberrant prion protein, which was clearlydistinguishable from a normal prion protein, was observed in a patientwho had developed the disease. At present, in addition to theaforementioned kuru disease and CJD, Gerstmann-Sträussler Syndrome(abbreviated to “GSS”) and fatal familial insomnia (abbreviated to“FFI”) are known as so-called prion diseases that result from prionaberrance in humans.

[0005] In the case of humans, a prion protein is composed of 253 aminoacids (SEQ ID NO: 2), which are bound to the surface of a cell membranethrough glycosyl-phosphatidyl-inositol (GPI) after a signal sequencecomprising 22 amino acids at the N terminus and 23 amino acids at the Cterminus has been removed (SEQ ID NO: 3).

[0006] Meanwhile, variations in the prion protein gene was discovered in1989, and familial GSS or familial CJD was found to develop with thevariation of only one amino acid of the prion protein. The prion proteinwas found to play an important role in CJD.

[0007] The aforementioned nvCJD was reported to develop early inpeople's lives (in their teens to 30's), and the deposition of theaberrant prion protein is observed not only in the central nervoussystem, which is known in other forms of CJD, but also in the folliculardendritic cell (FDC) in the lymphoid tissue, which was not observed inother forms of CJD (Hill A. F. et al., Lancet 1997, 349: 99-100). It isnot known approximately how many nvCJD carriers exist, and this is aserious issue of concern in Britain to such an extent that it isprohibited to supply blood products prepared from domestic blood.

[0008] For one of the reasons, the transmission (infection) of prionswithin the same species is easy, highly transmissible, and short in theincubation period while the transmission across species requires a longincubation period and is low rate of transmission. Because of thisacross-species barrier, it was very difficult to detect infectiousagents of human prion diseases such as CJD with using an animal model orthe like.

[0009] For example, an experiment regarding the transmission of thenvCJD to human type transgenic mice has been attempted (Hill, A. F. etal., Nature, Oct. 2,1997; 389 (6650): 448-50). However, it required along incubation period (228 days or longer) to develop any symptoms andwas not very successful (successful for only 25 out of 56 mice). It hasbeen recently reported that the nvCJD was transmitted in an incubationperiod of approximately 250 days with the use of bovine-type transgenicmice (Scott M. R. et al., Proc. Natl. Acad. Sci. USA 1999, 96:15137-42).

[0010] A system using transgenic animals has been reported as a methodfor detecting prions in a sample (JP Patent Publication (Kohyo) No.11-510496 A (1999)). In an example using mice, the development ofscrapie within approximately 50 days has been reported, however, it tookapproximately 200 days to develop into a human prion disease.

[0011] We demonstrated that an aberrant prion protein was deposited in asynapse in the central nervous system of a CJD patient and aCJD-infected mouse using a novel irnmunostaining procedure that isreferred to as the autoclave method (Kitamoto, T. et al., Am. J. Pathol.140: 1285-1294 (1992); Muramoto, T. et al., Am. J. Pathol. 140:1411-1420 (1992)). We also found that an aberrant prion protein wasdeposited in a cell other than one of the central nervous system, i.e.,the FDC (Kitamoto, T. et al., J. Virol. 65: 6292-6295 (1991)). Thedeposition of the aberrant prion protein in the FDC can be detected wellbefore a mouse develops the disease, and thus, a premorbid diagnosis canbe performed (Muramoto, T. et al., Am. J. Pathol. 140: 1411-1420 (1992);Muramoto, T. et al., Am. J. Pathol. 143: 1470-1479 (1993)).

[0012] Upon the intracranial administration of an infectious agent(mouse prion) to mice, they generally develop the disease in 120 to 140days. When the deposition of the aberrant prion protein in the FDC isemployed as an indication, detection of the infectious agent can becarried out in all the mice after 30 days post administration.

[0013] As described above, rapid determination of the prion infection inpharmaceuticals such as blood products requires early detection ofinfectious agents of human prion diseases such as CJD with using ananimal model, or the like.

[0014] The deposition of the aberrant prion protein in the FDC wasverified only in mice, and it could not be previously detected in humanCJD. Due to a species difference between humans and mice, it was knownthat the deposition of the aberrant prion protein in the FDC was notobserved in wild-type mice in the first inoculation for infection fromhuman CJD (Muramoto, T. et al., J. Virol. 67: 6808-6810 (1993)).

[0015] Accordingly, a method for directly detecting the aberrance in ahuman prion protein has been awaited.

DISCLOSURE OF THE INVENTION

[0016] Under the above circumstances, we have conducted concentratedstudies in which we prepared novel recombinant prion proteins derivedfrom human and mouse prion proteins and prepared transgenic animals andknock-in animals comprising the aforementioned proteins introducedtherein. As a result, we succeeded in developing a very effectivescreening method which can detect infectious agents of human ornon-human prion diseases in samples within an unprecedentedly shortperiod of time.

[0017] Specifically, the present invention provides the following (1) to(26).

[0018] (1) A screening method for infectious agents of human ornon-human prion diseases in a sample, which employs, as an indication,the deposition of an aberrant prion protein in the follicular dendriticcell (FDC) of a non-human animal.

[0019] (2) The screening method for according to (1) above, wherein thenon-human animal is a transgenic animal that expresses a humanized prionprotein gene.

[0020] (3) The screening method for according to (1) above, wherein thenon-human animal is a knock-in animal that expresses a humanized prionprotein gene.

[0021] (4) The screening method for according to any one of (1) to (3)above, wherein the sample is administered intraperitoneally,intracerebrally, intravascularly, or orally to the non-human animal.

[0022] (5) The screening method for according to any one of (1) to (4)above, wherein the deposition of the aberrant prion protein in the FDCis detected by a histological detection method, electrophoresis, and/orbinding assay.

[0023] (6) The screening method for according to any one of (1) to (5)above, wherein the deposition of the aberrant prion protein in the FDCis detected 14 to 700 days after the sample administration.

[0024] (7) The screening method for according to any one of (1) to (6)above, wherein the humanized prion protein is prepared by substituting apart of the exon 3 of the non-human animal prion protein gene with apart of the exon 3 of the human prion protein.

[0025] (8) The screening method for according to any one of (1) to (6)above, wherein the humanized prion protein is prepared by substituting 6residues on the C-terminal side among human-specific amino acid residuesin the human prion protein with corresponding amino acid residues in thenon-human animal prion protein that is used in the screening.

[0026] (9) The screening method for according to any one of (1) to (6)above, wherein the humanized prion protein comprises an amino acidsequence as shown in SEQ ID NO: 6 or 7.

[0027] (10) A recombinant humanized prion protein, which is encoded by arecombinant gene in which a part of the exon 3 of the non-human animalprion protein gene is substituted with a part of the exon 3 of the humanprion protein gene.

[0028] (11) A recombinant humanized prion protein, wherein 6 residues onthe C-terminal side among human-specific amino acid residues in thehuman prion protein are substituted with corresponding amino acids innon-human animal prion protein.

[0029] (12) A recombinant humanized prion protein, which comprises anamino acid sequence as shown in SEQ ID NO: 6 or 7.

[0030] (13) A gene or a fragment thereof, which encodes the proteinaccording to any one of (10) to (12) above.

[0031] (14) A gene or a fragment thereof, which comprises a nucleotidesequence as described in the following:

[0032] (a) the nucleotide sequence as shown in SEQ ID NO: 4;

[0033] (b) a nucleotide sequence that is a degenerate sequence of thenucleotide sequence as shown in SEQ ID NO: 4; or

[0034] (c) a nucleotide sequence that is hybridizable with the sequenceaccording to (a) or (b) under stringent conditions and encodes theprotein according to (10) above.

[0035] (15) A vector, which comprises the gene or a fragment thereofaccording to (13) or (14) above.

[0036] (16) A transgenic animal having the gene or a fragment thereofaccording to (13) or (14) above introduced therein.

[0037] (17) A knock-in animal having the gene or a fragment thereofaccording to (13) or (14) above introduced therein.

[0038] (18) A transgenic animal, which expresses a humanized prionprotein in the FDC.

[0039] (19) A knock-in animal, which expresses a humanized prion proteinin the FDC.

[0040] (20) A transgenic animal, which expresses the protein accordingto any one of (10) to (12) above in its brain and/or FDC.

[0041] (21) A knock-in animal, which expresses the protein according toany one of (10) to (12) above in its brain and/or FDC.

[0042] (22) A method for producing a transgenic animal, which expressesthe protein according to any one of (10) to (12) above.

[0043] (23) A method for producing a knock-in animal, which expressesthe protein according to any one of (10) to (12) above.

[0044] (24) The method for producing a knock-in animal according to (23)above comprising the following steps of:

[0045] (a) constructing a vector containing a non-human prion proteingene or a fragment thereof;

[0046] (b) substituting a part of the exon 3 of the non-human prionprotein gene with a part of the exon 3 of a human prion protein;

[0047] (c) inserting a loxp-surrounded antibiotic-resistant gene intothe 3′-non-translation region;

[0048] (d) introducing the resulting modified vector into the non-humanES cell;

[0049] (e) producing a chimera animal from a clone having homologousrecombination; and

[0050] (f) removing the antibiotic-resistant gene by introducing a Creenzyme-expressing plasmid into a fertilized egg of the F1 animal.

[0051] (25) A screening method for a preventive and/or therapeutic agentfor human or non-human prion diseases, which utilizes the transgenicanimal according to (18) or (20) above or the knock-in animal accordingto (19) or (21) above.

[0052] (26) A method, which utilizes the transgenic animal according to(18) or (20) above or the knock-in animal according to (19) or (21)above, for performing safety tests on various pharmaceuticals such asblood products derived from human or non-human animal organs, foods, orcosmetics.

[0053] This description includes part or all of the content as disclosedin the description and/or drawings of Japanese Patent Application No.2001-24279, which is a priority document of the present application.

BRIEF DESCRIPTION OF DRAWINGS

[0054]FIG. 1 is a diagram schematically showing the structure of therecombinant prion protein according to the present invention.

[0055]FIG. 2A shows the deposition of the aberrant prion protein in theFDC.

[0056]FIG. 2B shows immunostaining using an antibody that recognizes theC-terminus of a human-type prion protein.

[0057]FIG. 3 shows, by Western blotting, an infection with an aberrantprion protein in the spleen and in the brain.

[0058]FIG. 4 shows an embodiment of humanization by a vector used in thepreparation of the knock-in animals according to the present invention(knock-in vector), homologous recombination, or Cre-inducedrecombination.

[0059]FIG. 5 shows the structure of the transgenic vector used in thepresent invention.

[0060]FIG. 6 shows, by Western blotting, the expression of a recombinantprion protein in the spleen of the knock-in mouse and the transgenicmouse.

DESCRIPTION OF SEQUENCE LISTINGS

[0061] SEQ ID NO: 4 shows a nucleotide sequence of a chimeric priongene.

[0062] SEQ ID NO: 5 shows an amino acid sequence of a chimeric prionprotein.

[0063] SEQ ID NO: 6 shows an amino acid sequence of a ChM-type prionprotein.

[0064] SEQ ID NO: 7 shows an amino acid sequence of a ChV-type prionprotein.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0065] The present invention is hereafter described in more detail.

[0066] In this description, the term “non-human animal” refers to: amammalian animal such as a mouse, rat, hamster, guinea pig, rabbit, pig,cattle, sheep, cat, or dog; bird; or fish. In the present invention,non-human animals are not particularly limited, and a mouse isparticularly preferable from the viewpoints of breeding and handling.

[0067] In this description, the term “aberrant prion protein” refers toa prion protein, which has a conformation that is different from that ofa normal prion protein, is made insoluble by a surfactant, and is notpartially digested with protease. In human and animal prion diseases,this aberrant prion protein is always present in addition to a normalprion protein. This presence can be confirmed by, for example, Westernblotting after treatment with protease, detection of amyloid fibersconstituted by an aberrant prion protein using an electron microscopeafter the extraction of the protein, or detection by immunostainingutilizing the autoclave method, which we have developed (Shin, R. W. etal., Lab. Invest. 64: 693-702 (1991); Kitamoto, T. et al., J. Virol. 65:6292-6295 (1991); Kitamoto, T. et al., Am, J. Pathol. 140: 1285-1294(1992)).

[0068] The term “infectious agent” used herein refers to a factor thatis capable of developing the prion disease when administered to a humanor the transgenic animal or knock-in animal according to the presentinvention. Specific examples thereof include an aberrant prion proteinderived from human or non-human animals or a fragment thereof, and asubstance comprising the same. Examples of such samples comprisinginfectious agents include pharmaceuticals such as blood products derivedfrom human or non-human animals, foods, and cosmetics.

[0069] Further, the term “humanized prion protein” used herein refers toa prion protein in which a part thereof, which is originally expressedby its non-human animal host, is substituted with a sequence of a humanprion protein. Examples thereof include a recombinant humanized prionprotein that is encoded by a recombinant gene in which a part of theencoding region in a non-human animal prion protein gene has beensubstituted with a corresponding region of a human prion protein gene,and a recombinant humanized prion protein in which a part of theresidues in the human-specific amino acid residues of the human prionproten has been substituted with corresponding amino acid residues ofthe non-human animal prion protein.

[0070] An example of a humanized prion protein that is particularlypreferably used in the present invention is a recombinant humanizedprion protein, which is encoded by a recombinant gene in which a part ofthe exon 3 in the non-human animal prion protein gene is substitutedwith a part of the exon 3 in the human prion protein gene. Thisrecombinant humanized prion protein can be obtained in the followingmanner. A part of the exon 3 in the prion protein gene of the non-humananimal such as a mouse is substituted with a part of the exon 3 in thehuman prion protein gene by recombination to obtain a gene, and theresulting gene is incorporated into, for example, a vector to introduceit into a host to be expressed therein. The phrase “a part of the exon3” refers to a sequence that essentially comprises the region betweenthe SmaI site and the BstEII site in the prion protein translationregion of the exon 3. In the prion protein gene, a protein translationregion is present only in the exon 3.

[0071] Techniques of genetic engineering that are described in thisdescription such as gene recombination are commonly used in the art, andthose skilled in the art can suitably carry out them based on thedescription given herein.

[0072] Specifically, the humanized prion protein according to thepresent invention is expressed by a knock-in animal that is obtained by,for example, the following steps of:

[0073] (a) constructing a vector containing a non-human prion proteingene or a fragment thereof;

[0074] (b) substituting a part of the exon 3 of the non-human prionprotein gene with a part of the exon 3 of a human prion protein;

[0075] (c) inserting a loxp-surrounded antibiotic-resistant gene intothe 3′-non-translation region;

[0076] (d) introducing the resulting modified vector into the non-humanES cell;

[0077] (e) producing a chimera animal from a clone having homologousrecombination; and

[0078] (f) removing the antibiotic-resistant gene by introducing a Creenzyme-expressing plasmid into a fertilized egg of the F1 animal.

[0079] The humanized prion protein can be obtained by any techniqueknown to those skilled in the art such as site-directed mutagenesis orchemical synthesis as one in which, among amino acid residues that areknown to be human-specific, for example, 6 residues on the C-terminalside of the human prion protein are substituted with corresponding aminoacid residues of the non-human animal prion protein.

[0080] The base sequence of the human prion protein gene is known to bethe one as shown in SEQ ID NO: 1 and the amino acid sequence thereof isknown to be the total amino acid sequence as shown in SEQ ID NO: 2. Itbecomes a mature protein through processing with a signal sequencecomprising 22 amino acids at the N-terminus and an amino acid sequenceas shown in SEQ ID NO: 3 from which 23 amino acids at the C-terminushave been deleted. In SEQ ID NO: 3, residues 33, 34, 50, 58, 75, 87, 90,116, 121, 123, 133, 144, 146, 193, 197, 198, 205, 206, and 208 arepresumed to be human-specific (Kretzschmar, H. A. et al., DNA 1986, 5:315-24). We have made various studies concerning the substitution ofthese human-specific amino acids with corresponding amino acids ofnon-human animals. As a result, we found that a sequence in which 6residues on the C-terminal side are substituted with corresponding aminoacid residues of the non-human animal prion protein is particularlypreferably used in the screening method according to the presentinvention. An embodiment of this sequence is shown in SEQ ID NO: 6 or 7.SEQ ID NO: 6 is methionine at codon 129, and SEQ ID NO: 7 is valine atcodon 129. This polymorphism is observed in a normal human prion proteinand is also present in aberrant prion proteins that are observed invarious human prion diseases.

[0081] The present invention also provides a gene or a fragment thereofencoding the humanized prion protein according to the present inventionand a vector comprising the gene or a fragment thereof.

[0082] The gene encoding the humanized prion protein according to thepresent invention encodes a recombinant humanized prion proteincomprising an amino acid sequence as shown in SEQ ID NO: 6 or 7.Examples of other forms include a gene comprising a nucleotide sequenceas shown in SEQ ID NO: 4, a gene comprising a nucleotide sequence thatis a degenerate sequence of the nucleotide sequence as shown in SEQ IDNO: 4, and a gene comprising a nucleotide sequence that is hybridizablewith these sequences under stringent conditions. The stringentconditions used herein refer to a set of conditions under which aso-called specific hybrid is formed. For example, nucleotide sequencesthat are highly complementary to each other, i.e., nucleotide sequencesthat are at least 90%, and preferably at least 95% complementary to eachother hybridize with each other, but nucleotide sequences that are lesscomplementary do not hybridize under these conditions. Morespecifically, sodium concentration is 15 to 300 mM and preferably 15 to75 mM, and temperature is between 50 and 60° C. and preferably between55 and 60° C. A gene may be either DNA or RNA, and it may be obtained bysynthesis. A “fragment” refers to a part of the prion protein gene andpreferably comprises a nucleotide sequence that encodes the regionbetween codon 85 and codon 230 of SEQ ID NO: 2. Alternatively, afragment may be a defective type of this sequence, which refers to asequence comprising at least approximately 300 nucleotides.

[0083] Any vector comprising the gene or a fragment thereof may be usedas long as the gene can be expressed in a host animal cell into which ithas been introduced. The vector is not particularly limited as long asit is used in the art. An example thereof is a vector the expression ofwhich can be observed in any cell, which utilizes an actin promoter. Apromoter, enhancer, other regulator gene, or the like that can regulatethe expression of the gene can be suitably incorporated. Examples ofpromoters that can be preferably used in the present invention include apromoter that accelerates the expression in the FDC, such as a promoterof the CD21 (Cr2) gene. A reference can be made to literature, forexample, Zabel, M. D. et al., J. Immunol. Oct. 15, 2000; 165 (8):4437-45.

[0084] Meanwhile, the preparation of transgenic mice comprising a prionprotein gene introduced therein has been already reported (Telling G. C.et al., Cell Oct. 6, 1995, 6: 83 (1) 79-90). One example is a transgenicmouse, which comprises a completely human-type prion protein geneintroduced therein as shown in FIG. 1C. The incubation period thereinbefore the development of CJD was approximately 250 days. Anotherexample is a transgenic mouse, which comprises a protein gene in whichthe region between the Kpnl site and the BstEII site is a sequencederived from a human prion protein, and the N-terinus and the C-terminusare sequences derived from mouse prion proteins introduced therein asshown in FIG. 1A. The incubation period therein is approximately 200days. In contrast, the transgenic mouse according to the presentinvention comprises a protein gene in which the region between theN-terminus and the BstEII site is a sequence derived from a human prionprotein and the C-terminus is a sequence derived from a mouse prionprotein introduced therein as shown in FIG. 1B.

[0085] We prepared transgenic mice that comprise a gene (SEQ ID NO: 6 or7) encoding a protein that is schematically shown in FIG. 1B introducedtherein, and succeeded in obtaining mice, the incubation period thereinbefore the development of diseases is 147 days on average in the case ofhomozygous mice.

[0086] Accordingly, the present invention provides transgenic animalsthat express a recombinant humanized prion protein as shown in SEQ IDNO: 6 or 7. In the present invention, transgenic animals may behomozygous or heterozygous, and homozygous animals are more preferable.

[0087] However, it is unknown into which position and chromosome of thetransgenic animal the gene is incorporated. In order to obtain atransgenic animal having a short incubation period as described above,it has to be mated with a knockout animal of a prion protein gene. Wehave previously established a method for substituting genes using the EScell in which a mouse gene is substituted with a human type gene(Kitamoto, T. et al., Biochem. Biophys. Res. Commun. 222: 742-747(1996)). We improved this method and prepared knock-in mice in which apart of naturally occurring mouse gene was substituted with a human genehaving a sequence as shown in SEQ ID NO: 1. These knock-in mice had theincubation period of 151 days on average.

[0088] Specifically, the present invention provides knock-in animalsthat express the recombinant humanized prion protein as shown in SEQ IDNO: 6, 7, or 10. In the present invention, the knock-in animals may behomozygous or heterozygous, and homozygous animals are more preferable.The expression of recombinant humanized prion proteins are observed intheir whole bodies, and the expression can be detected particularly intheir brains and/or spleens.

[0089] An advantage of the method for substituting genes using the EScell is that it eliminates the need for mating with a knockout animal toremove a mouse endogenous prion protein, which was problematic in thecase of the transgenic animals. Also, the distribution and the level ofgene expression are similar to those of normal animals, and thus,completely natural expression can be realized.

[0090] When human or non-human animal-derived infectious agents areadministered to the model animals comprising humanized prion proteinsintroduced therein, it is presumed that the deposition of the aberrantprion protein is detected in the FDC. Accordingly, human-type transgenicanimals can be used for a method for rapidly screening infectious agentsthat employs the deposition of the aberrant prion protein in the FDC asan indication.

[0091] Based on the above presumption, we investigated the FDC after theresulting transgenic and knock-in mice developed the disease.Accordingly, aberrant prion proteins were not substantially detected inthe transgenic mice, however, the human-type aberrant prion proteinswere found to be deposited in the FDC of the spleen, lymph node, andintestinal lymphoid tissue (Peyer's patch) of the knock-in mice (FIG.2A).

[0092] Up to the present, it was impossible to determine whether thedeposition of the aberrant prion proteins in the FDC was caused byaggregation of aberrant prion proteins contained in the materials thatwere administered at the time of infection experiment or caused byconversion of normal prion proteins into aberrant prion proteins in theFDC. The C-terminus of the recombinant prion protein as shown in SEQ IDNO: 6 or 7 according to the present invention is of a mouse-type asdescribed above. Accordingly, we then prepared an antibody to theC-terminus of the human-type prion protein. The FDC was investigatedusing this antibody, and the FDC could not be stained (FIG. 2B). Thisindicates that the aberrant prion proteins deposited in the FDC were notsimple aggregation of human-type aberrant prion proteins contained inthe infectious agents.

[0093] We further examined the deposition of the aberrant prion proteinsin the spleen by Western blotting, which was already confirmed byimmunostaining. We confirmed the presence of the aberrant prion proteinin the spleen of the knock-in mouse (FIG. 3). Furthermore, theinfectivity in the spleen was confirmed by the direct infectionexperiment.(Table 2, Inoculation Experiment 3).

[0094] The knock-in mice that we prepared had an incubation period of151 days on average before the development of the disease. We examinedthe shortest period before the deposition of the aberrant prion proteinsin the FDC could be detected. As a result, it was found out thatdetection could be made in a very short time period of 14 days in theassay system using the knock-in mice according to the present invention(Table 6).

[0095] In contrast, the Ki-HuM mice that expressed complete human prionproteins (8 amino acids are deleted from SEQ ID NO: 10) required a verylong incubation period of 643 days, although all of them developed thedisease. In the immunohistostaining of the FDC in the spleen 75 daysafter the intraperitoneal inoculation, the rate of detection was jut 80%(4 out of 5 mice). As with the case of Ki-ChM, the deposition of theaberrant prion protein in the FDC was confirmed (Table 7). Specifically,with the use of the knock-in method in which the prion protein iseffectively expressed in the FDC, aberration of prion protein waseffectively observed in the FDC in the completely human-type animal tosome extent. In this type of animal, the aberration of the prion proteinis less likely to occur in the brain. Accordingly, with the use of theknock-in animals prepared by the method, which we claimed wherein theprion protein is effectively expressed in the FDC, it is possible todetect the deposition of the aberrant prion protein of non-human animalssuch as cattle in the FDC of the knock-in mouse, which expresses theprion protein of non-human animals such as cattle, as well as the prionprotein gene in which a specific region in the knock-in animal has beensubstituted.

[0096] The knock-in animal according to the present invention was foundto be unprecedentedly highly susceptible to prions as described above.Accordingly, the present invention also provides a biological assay,which employs the knock-in animal according to the present invention,and the deposition of the aberrant prion protein in the FDC thereof asan indication, thereby detecting the development of the human ornon-human animal prion diseases in any given samples. Ths assay can beused as a screening method for the infectious agents of the priondiseases in any given samples.

[0097] Examples of samples include various pharmaceuticals such as bloodor blood products derived from human or non-human animal organs, foods,and cosmetics. The sample is allowed to infect non-human animals,preferably transgenic animals that express humanized prion proteins, andparticularly preferably the knock-in animals according to the presentinvention. The sample may be administered intraperitoneally,intracerebrally, intravascularly, or orally. Intraperitonealadministration is preferable since a relatively large amount of samplecan be administered as described below. More specifically, for example,approximately 2 ml of a solution containing blood, organ, or apreparation derived thereform is administered to the knock-in animalsintraperitoneally to infect them.

[0098] After the infection, the deposition of the aberrant prion proteinin the FDC can be detected to determine whether the prion disease wasdeveloped or not using the sample. Any detection method may be used aslong as it can detect the deposition of the aberrant prion protein inthe FDC. Examples of detection methods include histological detection inwhich the deposition of the aberrant prion protein in the FDC of thePeyer's patch, which is a lymphoid tissue of the spleen, lymph node, orintestine is observed using an electron microscope, etc.,electrophoresis, and/or in situ hybridization, Western blotting, orELIZA in which the antibody to the aberrant prion protein is labeledwith a radioactive or nonradioactive label and binding assay isconducted. Detection may be carried out with the elapse of time afterthe infection. Alternatively, the period before the aberrant prionprotein is deposited in the FDC in the presence of the infectious agentsis previously determined by the control sample, and the deposition ofthe aberrant prion protein in the FDC may be detected after a determinedperiod has elapsed following sample infection, for example, 75 days. Theperiod of detection is not particularly limited as long as it is between14 days and 700 days after the administration. The deposition of theaberrant prion protein in the FDC can be employed as an indication todetermine the presence of the infectious agent within a significantlyshorter period of time than conventional methods.

[0099] Up to the present, no cases at all were reported in which theaberrant prion protein has been observed in the FDC of a mouse model ofhuman prion diseases, especially CJD. This is consistent with the factthat CJD is substantially sporadic and is not caused by exogenousinfection. nvCJD, which occurred in Britain in 1996, is considered to beassociated with the infectious agents derived from bovine spongifornencephalopathy and is classified as a transmissible prion disease.Accordingly, it is very critical to be able to detect the infectiousagent.

[0100] The present invention also provides a screening method whereinthe infectious agent is administered intraperitoneally, intracerebrally,intravascularly, or orally.

[0101] In the past, the infection experiments were mainly carried out byintracerebral administration. When a sample containing the infectiousagent is administered intracerebrally, the dose is limited. The samplecan be administered to the brain of a mouse only in an amount of 20 μlat the maximum, however, 2 ml of the sample can be administered to theabdominal cavity. In addition, the administration can be made severaltimes.

[0102] In a biological assay for general organs comprising blood inwhich the concentration of the infectious agents are considered to below, this quantitative difference of as much as 100 times is significantand affects the detection sensitivity. In the case of mice infected withCJD, for example, LD50 is 10⁻⁸/g in the brain and 10⁻³/g or lower in theblood according to infection experiments by intracerebraladministration. These results were obtained when 20 μl of the sample wasused for intracerebral administration. In contrast, when 100-fold amountof the sample were intraperitoneally administered, the concentration inthe organ (blood) having LD50 of 10⁻³/g was brought to the same level ashighly sensitive organs (such as the spleen) having LD50 correspondingto 10⁻⁵/g. Accordingly, intraperitoneal administration is a method withmuch promise as a future screening method for the infectious agents insamples.

[0103] Up to the present, no report has been made concerning thedevelopment of the disease upon intraperitoneal administration of theinfectious agents to a human-type transgenic mouse model. The data onthe transgenic mouse that we presented suggests the difficulty ofdeveloping the disease from the periphery by intraperitonealadministration without the deposition of the aberrant prion protein inthe FDC. In the SCID (deficient of T cell and B cell) mouse, whichdeveloped the disease by intracranial administration but does notdevelop the disease by intraperitoneal administration, the deposition ofthe aberrant prion protein in the FDC is not observed. This indicatesthat the aberrant prion protein is not transmitted to the brain withoutthe deposition thereof in the FDC.

[0104] In the knock-in mice according to the present invention, thehuman-type aberrant prion protein was deposited only after theadministration of the infectious agents. They were subjected tointraperitoneal administration. After an average of 283 days followingthe administration, all 11 mice had developed the diseases.Specifically, the infection of a human prion through the periphery wassuccessfully performed for the first time in the present invention(Table 5).

[0105] Further, the present invention provides a screening method forpreventive and/or therapeutic agents for human or non-human priondiseases, which utilizes the transgenic animal of the present inventionor the knock-in animal of the present invention.

[0106] As described above, the deposition of the aberrant prion proteinin the FDC can be detected within a short period after administration ofinfectious agent at high sensitivity with the use of the transgenicanimal or knock-in animal according to the present invention. Screeningcan be performed with this technique in which an agent, which is capableof blocking the deposition of the aberrant prion protein in the FDC orthe migration thereof from the FDC to the brain, can be administered totest animals before and after the administration of the infectious agentor simultaneously with the administration of the infectious agent. Thisenables the development of preventive and/or therapeutic agents forhuman or non-human prion diseases, which has been and is currentlyseriously problematic and any effective agent for which has not yet beenreported.

[0107] Prion diseases that are observed in various animals can beassayed using a knock-in mouse comprising the prion protein gene of theanimal of interest incorporated therein.

EXAMPLE Example 1 Preparation of Knock-In Vector

[0108] In order to prepare a recombinant human prion protein, anapproximately 10 kbp construct was selected as a vector for genesubstitution. This construct was centered on the exon 3 in which theregion between glycine (amino acid 40, nucleotide 118) and threonine(amino acid 187, nucleotide 561) in the translation region of the exon 3of the mouse prion protein gene (SEQ ID NO: 8 and SEQ ID NO: 9: aminoacid sequences) was substituted with a human prion protein gene. As aknock-in vector, a 3.5 kbp region between the BamHI site of the intron 2and the Smal site of the exon 3 of the mouse prion protein gene havingan intron as long as 20 kbp was used, the region between the SmaI siteand the BstEII site of the exon 3 was substituted with a human prionprotein gene, and the loxp-surrounded PGK-neo gene was inserted into theApaI site, which is a 3′-non-translation region of the exon 3 (FIG. 4).A mouse gene was used for the 3′ region, which was as large as 4 kbpbetween ApaI and EcoRV.

[0109] As a negative selection, the diphtheria toxin DTA gene wasinserted into the 3′ side, and a plasmid was linearized with NotI andthen introduced into the ES cell by electroporation. The ES cell wasanalyzed by Southern blotting by selecting 120 clones after G418selection (neomycin selection). Homologous recombination was observed in4 out of 120 analyzed clones. This was more effective compared to ourprevious experiment using loxP-neo-gpt-loxP in which a positive clonewas obtained at a rate of 1 out of 288 clones.

[0110] The efficiency of homologous recombination was further examinedusing the resulting knock-in vector. As a result, it was confirmed thatpositive clones could be efficiently obtained, i.e., at rates of 1 outof 60, 6 out of 180, 5 out of 175, and 2 out of 98 clones.

Example 2 Preparation of Knock-In Mouse

[0111] A gene was introduced into the ES cell of a mouse byelectroporation, and the G418-resistant clones were analyzed by Southernblotting. A positive clone (ES cell), in which homologous recombinationwas observed, was introduced into a morula to prepare chimera mice, andthe F1 mice were prepared by mating the chimera mice (germ-linepreparation). A Cre enzyme-expressing plasmid was introduced intofertilized eggs that were obtained by the mating of positive F1 mice,and the unnecessary neo gene was removed. Thus, the gene substitutionwith a human-type gene was completed.

[0112] The resulting heterozygous human-type knock-in mice (Ki-ChM) weremated with each other to prepare homozygous mice. The resulting knock-inmice expressed a recombinant prion protein as shown in SEQ ID NO: 6(Ki-ChM).

[0113] Similarly, knock-in mice that expressed a complete human prionprotein (SEQ ID NO: 10 comprising 4 repeat sequences, which should begenerally 5, of 8 amino acids,) were prepared as a control (Ki-HuM).

Example 3 Preparation of Transgenic Mice

[0114] Mouse prion protein genes were cloned from two types of mice, a129/SV mouse and an I/Ln mouse. Since the intron 2 of the 129/SV mouseis 20 kbp or longer, the gene of the I/Ln mouse with the shorter intron2 was also used. A 5 kbp region on the 5′ side of the 129/SV mouse, a 12kbp BamHI fragment containing the exons 1 and 2 of the I/Ln mouse, and a7 kbp exon 3 region of the 129/SV mouse were linked (FIG. 5). As withthe knock-in mouse in Example 2, the region between the SmaI site andthe BstEII site in the exon 3 was substituted with a human prionprotein. The thus prepared transgenic vector was cleaved out of theplasmid and directly introduced into the fertilized eggs of the BDF1mice. F0 mice, which successfully underwent the introduction, were matedto prepare F1 mice. The expression level was analyzed using this F1mouse, the F1 mouse was mated twice with a knockout mouse. Thus, amouse, which did not express a mouse prion protein but expressed only arecombinant prion protein (SEQ ID NO: 6 or 7) (Tg-ChM and Tg-ChV), wasprepared.

Example 4 Establishment of Infection by Intracerebral Administration toKnock-In Mice and Transgenic Mice

[0115] A brain emulsion was prepared from a frozen brain of a patient ofhuman sporadic CJD in a glass homogenizer to a concentration of 10%using a phosphate buffer (PBS). The right brain hemispheres of theknock-in mice (Ki-ChM and Ki-HuM) obtained in Example 2 and three typesof transgenic mice (Tg-ChM#30, Tg-ChV#21, and Tg-ChV#21) obtained inExample 3 were inoculated intracerebrally with the thus prepared brainemulsion under anesthesia using a 27-gauge syringe in amounts of 20 μleach. After the inoculation, neurological symptoms such as decreasedautonomic movement, atactic gait, abnormal gait, or tail rigidity wereobserved and developed. Individuals that developed emaciation ordebility were subjected to euthanasia and then to autopsy. It tookapproximately 21 days on average until the euthanasia following thedevelopment of the neurological symptom. The incubation period was theperiod between the day of inoculation, i.e., day 0, and the euthanasia.At the time of autopsy, mice were immobilized with buffered formalin,and some of the major organs were cryopreserved at −70° C.

[0116] All the mice were embedded in paraffin and then thinly sliced inthe laboratory to prepare pathological samples of prions only.Thereafter, the mice were subjected to HE staining andimmunohistostaining by the autoclave method, which we had devised, todiagnose the tissues based on the lesion and the deposition of theaberrant prion protein. Thus, the presence or absence of the priondisease was confirmed.

[0117] Regarding the Ki-ChM knock-in mice expressing a recombinant humanprion protein that were inoculated with human sporadic CJD (129M/M H3which was homozygous for methionine at codon 129, the incubation periodwas 151±6.7 days. The incubation periods in three types of transgenicmice (Tg-ChM#30, Tg-ChV#12, and Tg-ChV#21) that were inoculated with thesame material were 156±14.2 days, 175±15.3 days, and 192±4.0 days,respectively (Table 1). The incubation period was significantlyshortened compared to the incubation period in the Ki-HuM mice thatexpressed the complete human prion protein, i.e., 643±42.9 days. Incontrast, Wild (wild-type) mice into which no gene was introduced hadthe morbidity rate of only 36%, i.e., only 5 out of 14 examples, and theincubation period therein was 759±69.8 days.

[0118] The incubation period in the knock-in mice inoculated with humansporadic CJD (129V/M)-Su which was heterozygous for valine andmethionine at codon 129 was 141±5.3 days. The incubation periods inthree types of transgenic mice (Tg-ChM#30, Tg-ChV#12, and Tg-Ch#21),which were similarly inoculated with heterozygous sporadic CJD(129V/M)-Ph, were 154±20.8 days, 171±9.2 days, and 188±1.4 days,respectively (Table 1). TABLE 1 Establishment of infection byintracerebral administration of human prion Number of individualsIncubation developed the disease/ period Number of individuals MorbidityMouse Materials used for inoculation Days ± SD inoculated rate (%)Ki-ChM (Knock-in mouse) Sporadic CJD (129 M/M)-H3 151 ± 6.7 7/7 100Sporadic CJD (129 V/M)-Su 141 ± 5.3 5/5 100 Tg-ChM#30 (Transgenic mouse)Sporadic CJD (129 M/M)-H3  156 ± 14.2 11/11 100 Sporadic CJD (129V/M)-Ph  154 ± 20.8 5/5 100 Tg-ChV#12 (Transgenic mouse) Sporadic CJD(129 M/M)-H3  175 ± 15.3 18/18 100 Sporadic CJD (129 V/M)-Ph 171 ± 9.210/10 100 Tg-ChV#21 (Transgenic mouse) Sporadic CJD (129 M/M)-H3 192 ±4.0 3/3 100 Sporadic CJD (129 V/M)-Ph 188 ± 1.4 2/2 100 Ki-HuM (Knock-inmouse) Sporadic CJD (129 M/M)-H3  643 ± 42.9 4/4 100 Wild (Wild-typemouse) Sporadic CJD (129 M/M)-H3  759 ± 69.8  5/14* 36

[0119] Accordingly, the knock-in mice and the transgenic mice that wereobtained in Examples 2 and 3 according to the present invention werefound to be highly susceptible to human prions, and the infectivity ofhuman prion could be surely verified within a short period of time whichhad previously taken a long time and had been uncertain in conventionalwild-type mice. In particular, the knock-in mice were infected within anunprecedentedly short incubation period, i.e., approximately 150 days,with a human prion that was heterozygous for methionine or valine atcodon 129. This indicates that the knock-in mice according to thepresent invention can deal with the polymorphism of the human prionprotein gene. At the same time, the incubation period for the aboveinfection is equivalent to those among mice of mouse-adapted prions.Accordingly, it is considered that the “species barrier” in prioninfection was overcome. Further, the incubation period in the knock-inmice Ki-ChM was significantly shortened compared to the long incubationperiod in the completely human-type knock-in mice Ki-HuM. This indicatesthat the vector introduced and the recombinant human prion proteinexpressed thereby control the susceptibility to prion of these mice.

Example 5 Histological Detection of Aberrant Prion Protein in the FDC

[0120] The FDC was examined after the knock-in mice and the transgenicmice obtained in Examples 2 and 3 had developed the disease. As aresult, no aberrant prion protein was detected in the transgenic mice,although the deposition of the human-type aberrant prion protein wasobserved in the FDCs of the spleen, the lymph node, and the intestinallymphoid tissue (Peyer's patch) of the knock-in mice (FIG. 2A).

Example 6 Detection of Aberrant Prion Protein by Western Blotting

[0121] We also examined the deposition of the aberrant prion protein inthe spleen by Western blotting, which was already confirmed byimmunostaining. The presence of the aberrant prion protein was confirmedin the spleen of the knock-in mice.

[0122] The infected spleen, an uninfected spleen (a control), and theinfected brain of the knock-in mice obtained in Example 2 were analyzedby Western blotting.

[0123] When Western blotting is performed after treatment withproteinase K, it is known that three bands are formed from the aberrantprion protein. As shown in FIG. 3, the presence of the aberrant prionprotein was observed in the sample derived from the infected spleen andbrain since the three bands were observed. That is, a band containing nosugar chain attached from the bottom, a band containing a sugar chainattached through one site, and a band having sugar chains through twosites. In contrast, no band corresponding thereto was found in theuninfected sample. Compared to the brain, the level of the aberrantprion protein in the spleen was lower.

Example 7 Immunostaining Experiment on the FDC Using an Antibody to theC-Terminus of the Human-Type Prion Protein

[0124] A monoclonal antibody to the C-terminus of the human-type prionprotein was prepared. This antibody recognizes codons 215, 219, and 220of the human prion protein (SEQ ID NO: 2), and it does not react withthe amino acid sequences of the codons corresponding to those of themouse prion protein. Codons 215, 219, and 220 of the knock-in miceobtained in Example 2 (Ki-ChM) are substituted with those of mouse-typeinstead of human-type. Thus, this monoclonal antibody does not reacttherewith, and it reacts only with a completely human-type prionprotein. These knock-in mice were infected with a brain emulsion of thesporadic CJD example having a completely human-type aberrant prionprotein. If the FDC simply accumulates the injected completelyhuman-type aberrant prion protein, the FDC should be stained with thisantibody. In the experiment, however, the aberrant prion protein of theknock-in mice was not stained at all. That is, it is not a simpleaccumulation of the injected completely human-type prion proteins, but adeposition, in the FDC, of the aberrated human-type prion protein of theknock-in mice having mouse-type C-terminuses.

Example 8 Comparison Between Transgenic Mice and Knock-In Mice

[0125] In order to examine the reason why the deposition of the aberrantprion protein was detected in the FDC of knock-in mice but not intransgenic mice in Example 5, the expression of the recombinant prionprotein in the spleen was assayed by Western blotting.

[0126] The expression level of the normal prion protein in the spleen ofthe transgenic mouse (Tg-ChV#12), which expresses twice as manyrecombinant prion proteins as those expressed in the brain of theknock-in mouse (Ki-ChM), was examined by Western blotting. The resultsare shown in FIG. 6. In FIG. 6, “a” to “d” represent knock-in mice and“e” represents a transgenic mouse. “d” and “e” are electrophoresedfractions of the spleens of the same tissue weight, “c” is a 50%electrophoresed fraction, “b” is a 25% electrophoresed fraction, and “a”is a 12.5% electrophoresed fraction by tissue weight based on “d.” Theintensity of the immune response of “e” is considered to besubstantially equivalent to that of “b” and the expression level in thetransgenic mice was approximately 25% of that in the knock-in mice.

[0127] This demonstrates that the distribution of expression in thetransgenic mice is different from that in the wild-type mice.Specifically, the expression level in the transgenic mice is merelyextremely low, and this does not mean that the recombinant prion proteinis not expressed in the spleen (FDC). Accordingly, it is considered thatthe expression in the spleen (FDC) of the transgenic mice was determinedto be negative.

[0128] Thus, the deposition of the aberrant prion protein in the FDC wasfound to be detectable with the use of the transgenic animals accordingto the present invention, if a suitable detection method was employed.

Example 9 Confirmation of Infectivity of Mice That Developed the DiseaseAfter Being Infected With Human Prion

[0129] In the direct infection experiment, the infectivity of theaberrant prion proteins that were found in the brain and the spleen ofthe mice were verified.

[0130] The knock-in mice (Ki-ChM) obtained in Example 2 were inoculatedintracerebrally with a 10% brain emulsion prepared from a sporadic CJDexample. A brain emulsion was prepared from the brains and the spleensof the knock-in mice (Ki-ChM), which developed the disease, in a glasshomogenizer to a concentration of 10% using a phosphate buffer (PBS).The right brain hemispheres of the knock-in mice (Ki-ChM) wereinoculated intracerebrally with the brain emulsion under anesthesiausing a 27-gauge syringe in amounts of 20 μl each. As with theconventional methods that were carried out in all the mouse inoculationexperiments in Examples herein, neurological symptoms such as decreasedautonomic movement, atactic gait, abnormal gait, or tail rigidity wereobserved and developed after the inoculation. Individuals that developedemaciation or debility were subjected to euthanasia and then to autopsy.

[0131] As a result, 6 out of 6 mice (100%), which were inoculated with abrain emulsion of mice that had developed the disease, developed thedisease, and the incubation period was 123±10.0 days. In contrast, 5 outof 5 mice (100%), which were inoculated with a spleen emulsion of themice that had developed the disease, developed the disease, and theincubation period was 156±7.9 days. As is apparent from these results,the presence of the infectivity as well as the deposition of theaberrant protein were confirmed in the brain and the spleen (FDC) of theknock-in mice (Ki-ChM) that had developed the diseased after theinoculation of a human brain emulsion (Table 2). TABLE 2 Confirmation ofinfectivity of mice that developed the disease after being infected withhuman prion Number of individuals that developed the Incubationdisease/number of Materials used Inoculated period individuals forinoculation mouse (days ± SD) inoculated Inoculation experiment 1,Ki-ChM 151 ± 6.7 7/7 Human sporadic CJD, 10% brain emulsion Inoculationexperiment 2, Ki-ChM  123 ± 10.0 6/6 10% brain emulsion of mouse thatdeveloped the disease (Inoculation Experiment 1) Inoculation experiment3, Ki-ChM 156 ± 7.9 5/5 10% spleen emulsion of mouse that developed thedisease (Inoculation Experiment 1)

Example 10 Experiment for Detecting Aberrant Prion of CJD Patient in theFDC

[0132] A brain emulsion was prepared from the frozen brain of a CJDpatient in a glass homogenizer to a concentration of 10% using aphosphate buffer (PBS). The knock-in mice (Ki-ChM) were inoculatedintraperitoneally with the brain emulsion using a 26-gauze syringe inamounts of 50 μl each. Mice were subjected to euthanasia at 75 daysafter the inoculation and then to autopsy. At the time of autopsy, micewere immobilized with buffered formalin, and some of the major organswere cryopreserved at −70° C. All the mice were embedded in paraffin andthen thinly sliced in the laboratory to prepare pathological samples ofprions only. Thereafter, the mice were subjected to HE staining andimmunohistostaining by the autoclave method, which we had devised, toexamine the deposition of the aberrant prion protein in the FDC.

[0133] As a result, the aberrant prion proteins were detected in theFDCs of all the Ki-ChM mice inoculated with a human prion derived fromeither human sporadic CJD (129 M/M)-H3 which was homozygous formethionine at codon 129, human sporadic CJD (129 V/M)Su which washeterozygous for valine and methionine at codon 129, or human CJD bydura mater transplantation, CJD-TMD-Du/c. Specifically, the detectionrate was 100% (Table 3). TABLE 3 Detection of aberrant prion of CJDpatient in the FDC Number of positive Number of days FDC/NumberInoculated following the of examined Detection Material used forinoculation mice inoculation individuals rate Sporadic CJD (129 M/M)-H3,10% human Ki-ChM 75 5/5 100% brain emulsion Sporadic CJD (129 V/M)-Su,10% human Ki-ChM 75 5/5 100% brain emulsion Dura mater transplantedCJD-TMD-Du/C, Ki-ChM 75 6/6 100% 10% human brain emulsion

Example 11 CJD Infection by Dura Mater Transplantation

[0134] A brain emulsion was prepared from the frozen brain of a patientwho was definitely diagnosed to have developed CJD after a dura matertransplantation (CJD-TMD-Du/c) in a glass homogenizer to a concentrationof 10% using a phosphate buffer (PBS). The knock-in mice (Ki-ChM) wereinoculated intraperitoneally with the brain emulsion using a 26-gauzesyringe in amounts of 50 μl each. Mice were subjected to euthanasia at75 days after the inoculation and then to autopsy. At the time ofautopsy, mice were immobilized with buffered formalin, and some of themajor organs were cryopreserved at −70° C. All the mice were embedded inparaffin and then thinly sliced in the laboratory to preparepathological samples of prions only. Thereafter, the mice were subjectedto HE staining and immunohistostaining by the autoclave method, which wehad devised, to examine the deposition of the aberrant prion protein inthe FDC.

[0135] As a result, the aberrant prion proteins were detected in thespleen FDCs of all the 5 inoculated Ki-ChM mice (Table 3).

Example 12 Detection of Aberrant Prion Derived From British nvCJDPatient

[0136] A brain emulsion was prepared from the frozen brain of a patientof new variant CJD (hereinafter abbreviated to “nvCJD”), which waspresumed to have derived from BSE in Britain, in a glass homogenizer toa concentration of 10% using a phosphate buffer (PBS). In order toremove infectious agents other than prions, the brain emulsion wasmaintained at 60° C. for 30 minutes. Thereafter, the emulsion wascryopreserved at −70° C. until the inoculation. At the time ofinoculation, the emulsion was defrosted, and the knock-in mice (Ki-ChM)were inoculated intraperitoneally therewith using a 26-gauze syringe inamounts of 50 μl each. They were subjected to euthanasia at 75 daysafter the inoculation and then to autopsy. At the time of autopsy, micewere immobilized with buffered formalin, and some of the major organswere cryopreserved at −70° C. All the mice were embedded in paraffin andthen thinly sliced in the laboratory to prepare pathological samples ofprions only. Thereafter, the mice were subjected to HE staining andimmunohistostaining by the autoclave method, which we had devised, toexamine the deposition of the aberrant prion protein in the FDC.

[0137] As a result, aberrant prion proteins were detected at thedetection rate of 100%. Specifically, 5 out of 5, 4 out of 4, and 4 outof 4 of the three cases of nvCJD patients' brains, i.e., nv-96/02,nv-96/07, and nv-96/45, respectively.

[0138] A novel biological assay method using the FDC of the knock-inmice (Ki-ChM) according to the present invention was also useful for thediagnosis of prions derived from nvCJD patients in Britain (Table 4).TABLE 4 Detection of aberrant prion of British nvCJD patient Number ofNumber of positive Material used Inoculated days from FDC/Number ofDetection for inoculation mice the inoculation examined individuals rateBritish nvCJD human Ki-ChM 75 5/5 100% 10% brain emulsion, nv-96/02British nvCJD human Ki-ChM 75 4/4 100% 10% brain emulsion, nv-96/07British nvCJD human Ki-ChM 75 4/4 100% 10% brain emulsion, nv-96/45

Example 13 Examination of Assay System 1 (Intracerebral andIntraperitoneal Administrations)

[0139] A brain emulsion was prepared from the frozen brain of a humanCJD patient in a glass homogenizer to a concentration of 10% using aphosphate buffer (PBS). In order to remove infectious agents other thanprions, the brain emulsion was maintained at 60° C. for 30 minutes.Thereafter, the emulsion was cryopreserved at −70° C. until theinoculation. At the time of inoculation, the emulsion was defrosted, andthe right brain hemispheres of the knock-in mice (Ki-ChM) obtained inExample 2 were inoculated intracerebrally therewith under anesthesiausing a 27-gauge syringe in amounts of 20 μl each. After theinoculation, neurological symptoms such as decreased autonomic movement,atactic gait, abnormal gait, or tail rigidity were observed anddeveloped. Individuals that developed emaciation or debility weresubjected to euthanasia and then to autopsy. It took approximately 21days on average until the euthanasia following the development of theneurological symptom. The incubation period was the period between theday of inoculation, i.e., day 0, and the euthanasia. At the time ofautopsy, mice were immobilized with buffered formalin, and some of themajor organs were cryopreserved at −70° C. All the mice were embedded inparaffin and then thinly sliced in the laboratory to preparepathological samples of prions only. Thereafter, the mice were subjectedto HE staining and immunohistostaining by the autoclave method, which wehad devised, to diagnose the tissues based on the lesion and thedeposition of the aberrant prion protein. Thus, the presence or absenceof the prion disease was confirmed. TABLE 5 Examination of assay system1 (intracerebral and intraperitoneal administrations) Number ofindividuals that Inoculated Inoculation Incubation period developed thedisease/number of Material used for inoculation mouse route (days ± SD)individuals inoculated Sporadic CJD (129M/M), human Ki-ChM Intracerebral151 ± 6.7 7/7 10% brain emulsion-H3 Sporadic CJD (129M/M), human Ki-ChMIntraperitoneal 283 ± 9.2 11/11 10% brain emulsion-H3 Sporadic CJD(129V/M), human Ki-ChM Intracerebral 141 ± 5.3 5/5 10% brain emulsion-SuSporadic CJD (129 V/M, Ki-ChM Intracerebral 177 ± 4.9 4/4 M232R), human10% brain emulsion, TMD-232 CJD after dura mater Ki-ChM Intracerebral 167 ± 24.7 6/6 transplantation, human 10% brain emulsion, TMD-Du/C

[0140] As a result, the incubation period in the knock-in nice that wereinoculated with human sporadic CJD (129 M/M)-H3 which was homozygous formethionine at codon 129 was 151±6.7 days. The incubation period wasextended in the case of intraperitoneal inoculation. However, all 11mice developed the disease in 283±9.2 days. The incubation period in theknock-in mice that were inoculated with human sporadic CJD (129 V/M)-Suwhich was heterozygous for valine and methionine at codon 129 was141±5.3 days. In the case of a human brain emulsion that washeterozygous and had gene mutation in which methionine at codon 232 hadbeen substituted with arginine, the incubation period was somewhatextended, i.e., 177±4.9 days, and all the mice developed the disease.Mice, which were inoculated with a 10% brain emulsion of a humanaffected with CJD after dura mater transplantation, TMD-Du/C, developedthe disease in the incubation period of 167±24.7 days.

[0141] Accordingly, the knock-in mice of the present invention, Ki-ChM,were found to be highly susceptible to various types of human prions.Since they are susceptible through intraperitoneal inoculation as wellas intracerebral inoculation, they are presumed to be susceptible tohuman prion infection through various peripheries.

Example 14 Examination of Assay System 2—Observation With the Elapse ofTime

[0142] A brain emulsion was prepared from a frozen brain of a human CJDpatient in a glass homogenizer to a concentration of 10% using aphosphate buffer (PBS). In order to remove infectious agents other thanprions, the brain emulsion was maintained at 60° C. for 30 minutes.Thereafter, the emulsion was cryopreserved at −70° C. until theinoculation. At the time of inoculation, the emulsion was defrosted, andthe knock-in mice (Ki-ChM) obtained in Example 2 were then inoculatedintraperitoneally therewith using a 26-gauge syringe in amounts of 50 pleach. Mice were subjected to euthanasia 14 days, 31 days, 44 days, 60days, 75 days, or 150 days after the inoculation. At the time ofautopsy, mice were immobilized with buffered formalin, and some of themajor organs were cryopreserved at −70° C. All the mice were embedded inparaffin and then thinly sliced in the laboratory to preparepathological samples of prions only. Thereafter, the mice were subjectedto HE staining and immunohistostaining by the autoclave method, which wehad devised, to detect the lesion and the aberrant prion protein.

[0143] As a result, the deposition of the aberrant prion proteins in theFDCs of 2 out of 4 knock-in mice (Ki-ChM) (50%) 14 days after theinoculation were observed by immunohistostaining. Thereafter, theaberrant prion proteins were present in the FDCs of all the inoculatedmice from day 31 to day 150. Abnormal prion proteins or otherhistopathological changes, however, were not detected in the targetorgan, the central nervous system, from day 14 to day 150.

[0144] According to the FDC-based biological assay using the knock-inmice (Ki-ChM) of the present invention, aberrant prion proteins can bedetected within a very short time period of 14 days. This indicates thatdetection can be continuously performed (Table 6). TABLE 6 Detection ofaberrant prion protein in the FDC of Ki-ChM mice with the elapse of timeNumber of days Number of after the Positive FDC/number administration ofmice examined Detection rate 14 days 2/4  50% 31 days 6/6 100% 44 days7/7 100% 60 days 6/6 100% 75 days 5/5 100% 150 days  7/7 100%

Example 15 Examination of Assay System 3—Examination of Concentration

[0145] In Example 14, the deposition of the aberrant prion proteins wasfound to be detectable in the FDCs of the knock-in mice (Ki-ChM)according to the present invention within at least 14 days using a 10%brain emulsion of human sporadic CJD. Subsequently, the lowestdetectable concentration was examined by diluting the material forinoculation.

[0146] The aforementioned 10% frozen brain emulsion (tenfold diluted) ofa sporadic CJD-affected human was defrosted, and a 1% (100-folddiluted), 0.1% (1000-fold diluted,) and 0.01% (10000-fold diluted)solutions were prepared using the same PBS. The knock-in mice (Ki-ChM)that were obtained in Example 2 were inoculated intraperitoneally withthe brain emulsion using a 26-gauze syringe in amounts of 50 μl each.Mice were subjected to euthanasia at 75 days after the inoculation andthen immobilized on buffered formalin. All the samples were embedded inparaffin and then thinly sliced in the laboratory to preparepathological samples of prions only. Thereafter, they were subjected toimmunohistostaining by the autoclave method, which we had devised, todetect the deposition of the aberrant prion protein.

[0147] As a result, aberrant prion proteins were detected in the FDCs of80% or more individuals inoculated with a 0.01% (10,000-fold diluted)solution.

[0148] This indicates that the use of the FDC of the knock-in mice(Ki-ChM) of the present invention enables the detection of infectivityof sporadic CJD-infected human brain even if it is diluted 10,000-foldor more.

Example 16 Comparison of Susceptibility Between Knock-In Mice Ki-ChM andKi-HuM

[0149] Susceptibility to prion by intracerebral inoculation and that byintraperitoneal inoculation were compared using two types of knock-inmice, Ki-ChM and Ki-Hun, which were obtained in Example 2. Specifically,the right brain hemisphere of the knock-in mice (Ki-ChM and Ki-HuM)obtained in Example 2 were inoculated intracerebrally with a 10% brainemulsion of the sporadic CJD (129 M/M)-H3 example using a 27-gaugesyringe in amounts of 20 μl each. Also, 50 μl each of the brain emulsionwas injected intraperitoneally using a 26-gauze syringe.

[0150] In the case of intracerebral inoculation, neurological symptomssuch as decreased autonomic movement, atactic gait, abnormal gait, ortail rigidity were observed and developed after the inoculation, as withthe conventional methods that were carried out in all the mouseinoculation experiments in Examples herein. Individuals that developedemaciation or debility were subjected to euthanasia and then to autopsy.The incubation period was the period between the day of inoculation,i.e., day 0, and the euthanasia. At the time of autopsy, mice wereimmobilized with buffered formalin, and some of the major organs werecryopreserved at −70° C.

[0151] In the case of intraperitoneal inoculation, mice were subjectedto euthanasia 75 days after the inoculation and then to autopsy. At thetime of autopsy, they were immobilized with buffered formalin. Some ofthe major organs were cryopreserved at −70° C. All the mice wereembedded in paraffin and then thinly sliced in the laboratory to preparepathological samples of prions only. Thereafter, the mice were subjectedto HE staining and immunohistostaiing by the autoclave method, which wehad devised, to detect the deposition of the aberrant prion protein inthe FDC.

[0152] Consequently, all the human-type knock-in mice Ki-ChM developedthe disease within the incubation period of 151±6.7 days. Although allthe knock-in mice (Ki-HuM) that expressed complete human prion proteins(SEQ ID NO: 10 comprising 4 repeat sequences, which should be generally5, of 8 amino acids) developed the disease, it required a very longincubation period of 643 ±42.9 days. According to immunohistostaining ofthe spleen FDC 75 days after the intraperitoneal inoculation, aberrantprion proteins were detected at 100% (5 out of 5 mice) in the case ofKi-ChM. The detection rate, however, was just 80% (4 out of 5 mice) inthe case of Ki-HuM (Table 7). TABLE 7 Comparison of susceptibilitybetween knock-in mice Ki-ChM and Ki-HuM Lineage of mice Inoculationroute Ki-ChM Ki-HuM Intracerebral inoculation Incubation period 151 ±6.7 643 ± 42.9 Number of crisis 7/7 (100%) 4/4 (100%) IntraperitonealPositive FDC 5/5 (100%) 4/5 (100%) inoculation (75 days later)

[0153] In consideration of both the incubation period in the case ofintracerebral inoculation and the detection rate of aberrant prionproteins in the FDC 75 days later, aberrant prion proteins were found tobe always deposited in the FDCs of the knock-in mice regardless of theirconstructs. Specifically, the use of the knock-in method, wherein theprion proteins are effectively expressed in the FDC, enables effectiveobservation of aberration in the FDC to some extent in the case ofcompletely hunan-type animals, wherein aberration of prion protein isunlikely to occur in brains. At the same time, even in a lineage havinga long incubation period, i.e., a completely human-type animal in whichaberration is unlikely to occur in the brain, the probability ofaberrant prion protein deposition in the FDC does not reach 100%. Thismeans that the deposition of the aberrant prion protein in the FDCreflects the susceptibility of mice to human prions. Also, it reflectsthe fact that the aberration of completely human-type prion proteins ishighly unlikely to occur.

Industrial Applicability

[0154] As described above, the present invention enabled the preparationof an animal model that is unprecedentedly highly susceptible to humanprion proteins. With the use of this animal model, the present inventioncan provide a novel screening method that can be used in safety tests ofhuman or non-human animal prion diseases.

[0155] The knock-in animal that is obtained by the present invention isexcellent for use in an assay system for nvCJD aberrant prion proteins.In particular, in the case of intraperitoneal administration, the amountof infectious agents used for inoculation can be raised to 100 timescompared with that of intracerebral administration. The infectivity ofblood, which is considered to be low, can be investigated byadministering a large amount thereof.

[0156] Accordingly, the knock-in animal of the present invention will beessential for final safety tests of preparations that are produced fromblood or organs of human or non-human animals. nvCJD is considered toinfect as follows. The aberrant prion protein is first deposited in thetonsilla and the FDC of lymphoid tissues of digestive organs byingesting bovine prion proteins. The aberrant prion protein is thentransported from the FDC to the brain. As with the case of this nvCJD,the knock-in animal obtained by the present invention was found todevelop the disease through the deposition of the aberrant prion proteinin the FDC through peripheral routes, followed by transportation of theaberrant prion protein to the brain. This was verified through theirinfection with human prions. Accordingly, this model can be effectivenot only in the establishment of rapid biological assay but also in ascreening system for developing medicines to block the aberrant prionproteins from being transported from the FDC to the brain in the future.

[0157] All publications, patents, and patent applications cited hereinare incorporated herein by reference in their entirety.

1 10 1 762 DNA Homo sapiens CDS (1)..(762) 1 atg gcg aac ctt ggc tgc tggatg ctg gtt ctc ttt gtg gcc aca tgg 48 Met Ala Asn Leu Gly Cys Trp MetLeu Val Leu Phe Val Ala Thr Trp 1 5 10 15 agt gac ctg ggc ctc tgc aagaag cgc ccg aag cct gga gga tgg aac 96 Ser Asp Leu Gly Leu Cys Lys LysArg Pro Lys Pro Gly Gly Trp Asn 20 25 30 act ggg ggc agc cga tac ccg gggcag ggc agc cct gga ggc aac cgc 144 Thr Gly Gly Ser Arg Tyr Pro Gly GlnGly Ser Pro Gly Gly Asn Arg 35 40 45 tac cca cct cag ggc ggt ggt ggc tggggg cag cct cat ggt ggt ggc 192 Tyr Pro Pro Gln Gly Gly Gly Gly Trp GlyGln Pro His Gly Gly Gly 50 55 60 tgg ggg cag cct cat ggt ggt ggc tgg gggcag ccc cat ggt ggt ggc 240 Trp Gly Gln Pro His Gly Gly Gly Trp Gly GlnPro His Gly Gly Gly 65 70 75 80 tgg gga cag cct cat ggt ggt ggc tgg ggtcaa gga ggt ggc acc cac 288 Trp Gly Gln Pro His Gly Gly Gly Trp Gly GlnGly Gly Gly Thr His 85 90 95 agt cag tgg aac aag ccg agt aag cca aaa accaac atg aag cac atg 336 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr AsnMet Lys His Met 100 105 110 gct ggt gct gca gca gct ggg gca gtg gtg gggggc ctt ggc ggc tac 384 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly GlyLeu Gly Gly Tyr 115 120 125 atg ctg gga agt gcc atg agc agg ccc atc atacat ttc ggc agt gac 432 Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile HisPhe Gly Ser Asp 130 135 140 tat gag gac cgt tac tat cgt gaa aac atg caccgt tac ccc aac caa 480 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His ArgTyr Pro Asn Gln 145 150 155 160 gtg tac tac agg ccc atg gat gag tac agcaac cag aac aac ttt gtg 528 Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser AsnGln Asn Asn Phe Val 165 170 175 cac gac tgc gtc aat atc aca atc aag cagcac acg gtc acc aca acc 576 His Asp Cys Val Asn Ile Thr Ile Lys Gln HisThr Val Thr Thr Thr 180 185 190 acc aag ggg gag aac ttc acc gag acc gacgtt aag atg atg gag cgc 624 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp ValLys Met Met Glu Arg 195 200 205 gtg gtt gag cag atg tgt atc acc cag tacgag agg gaa tct cag gcc 672 Val Val Glu Gln Met Cys Ile Thr Gln Tyr GluArg Glu Ser Gln Ala 210 215 220 tat tac cag aga gga tcg agc atg gtc ctcttc tcc tct cca cct gtg 720 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu PheSer Ser Pro Pro Val 225 230 235 240 atc ctc ctg atc tct ttc ctc atc ttcctg ata gtg gga tga 762 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile ValGly 245 250 2 253 PRT Homo sapiens 2 Met Ala Asn Leu Gly Cys Trp Met LeuVal Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys LysArg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro GlyGln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly GlyGly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro SerLys Pro Lys Thr Asn Met Lys His Met 100 105 110 Ala Gly Ala Ala Ala AlaGly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 Met Leu Gly Ser AlaMet Ser Arg Pro Ile Ile His Phe Gly Ser Asp 130 135 140 Tyr Glu Asp ArgTyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 Val TyrTyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175 HisAsp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245250 3 208 PRT Homo sapiens 3 Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn ThrGly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser Pro Gly Gly Asn ArgTyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp Gly Gln Pro His Gly Gly GlyTrp Gly Gln Pro His Gly 35 40 45 Gly Gly Trp Gly Gln Pro His Gly Gly GlyTrp Gly Gln Pro His Gly 50 55 60 Gly Gly Trp Gly Gln Gly Gly Gly Thr HisSer Gln Trp Asn Lys Pro 65 70 75 80 Ser Lys Pro Lys Thr Asn Met Lys HisMet Ala Gly Ala Ala Ala Ala 85 90 95 Gly Ala Val Val Gly Gly Leu Gly GlyTyr Met Leu Gly Ser Ala Met 100 105 110 Ser Arg Pro Ile Ile His Phe GlySer Asp Tyr Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu Asn Met His Arg TyrPro Asn Gln Val Tyr Tyr Arg Pro Met 130 135 140 Asp Glu Tyr Ser Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160 Thr Ile Lys GlnHis Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170 175 Thr Glu ThrAsp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180 185 190 Ile ThrGln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly Ser 195 200 205 4768 DNA Artificial Sequence Description of Artificial SequenceChimera-type prion gene 4 atg gcg aac ctt ggc tac tgg ctg ctg gcc ctcttt gtg act atg tgg 48 Met Ala Asn Leu Gly Tyr Trp Leu Leu Ala Leu PheVal Thr Met Trp 1 5 10 15 act gat gtc ggc ctc tgc aaa aag cgg cca aagcct gga ggg tgg aac 96 Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys ProGly Gly Trp Asn 20 25 30 acc ggt gga agc cgg tat ccc ggg cag ggc agc cctgga ggc aac cgc 144 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro GlyGly Asn Arg 35 40 45 tac cca cct cag ggc ggt ggt ggc tgg ggg cag cct catggt ggt ggc 192 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His GlyGly Gly 50 55 60 tgg ggg cag cct cat ggt ggt ggc tgg ggg cag ccc cat ggtggt ggc 240 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly GlyGly 65 70 75 80 tgg gga cag cct cat ggt ggt ggc tgg ggt caa gga ggt ggcacc cac 288 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly ThrHis 85 90 95 agt cag tgg aac aag ccg agt aag cca aaa acc aac atg aag cacatg 336 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met100 105 110 gct ggt gct gca gca gct ggg gca gtg gtg ggg ggc ctt ggc ggctac 384 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr115 120 125 rtg ctg gga agt gcc atg agc agg ccc atc ata cat ttc ggc agtgac 432 Xaa Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp130 135 140 tat gag gac cgt tac tat cgt gaa aac atg cac cgt tac ccc aaccaa 480 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln145 150 155 160 gtg tac tac agg ccc atg gat gag tac agc aac cag aac aacttt gtg 528 Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn PheVal 165 170 175 cac gac tgc gtc aat atc aca atc aag cag cac acg gtc accacc acc 576 His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr ThrThr 180 185 190 acc aag ggg gag aac ttc acc gag acc gat gtg aag atg atggag cgc 624 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met GluArg 195 200 205 gtg gtg gag cag atg tgc gtc acc cag tac cag aag gag tcccag gcc 672 Val Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu Ser GlnAla 210 215 220 tat tac gac ggg aga aga tcc agc agc acc gtg ctt ttc tcctcc cct 720 Tyr Tyr Asp Gly Arg Arg Ser Ser Ser Thr Val Leu Phe Ser SerPro 225 230 235 240 cct gtc atc ctc ctc atc tcc ttc ctc atc ttc ctg atcgtg gga tga 768 Pro Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile ValGly 245 250 255 5 255 PRT Artificial Sequence Description of ArtificialSequence Chimera-type prion protein 5 Met Ala Asn Leu Gly Tyr Trp LeuLeu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr Asp Val Gly Leu Cys LysLys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr ProGly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His GlyGly Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys ProSer Lys Pro Lys Thr Asn Met Lys His Met 100 105 110 Ala Gly Ala Ala AlaAla Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 Xaa Leu Gly SerAla Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp 130 135 140 Tyr Glu AspArg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 ValTyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195200 205 Val Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu Ser Gln Ala210 215 220 Tyr Tyr Asp Gly Arg Arg Ser Ser Ser Thr Val Leu Phe Ser SerPro 225 230 235 240 Pro Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu IleVal Gly 245 250 255 6 209 PRT Artificial Sequence Description ofArtificial Sequence ChM-type prion protein 6 Lys Lys Arg Pro Lys Pro GlyGly Trp Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser ProGly Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp Gly Gln ProHis Gly Gly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly Gly Trp Gly Gln ProHis Gly Gly Gly Trp Gly Gln Pro His Gly 50 55 60 Gly Gly Trp Gly Gln GlyGly Gly Thr His Ser Gln Trp Asn Lys Pro 65 70 75 80 Ser Lys Pro Lys ThrAsn Met Lys His Met Ala Gly Ala Ala Ala Ala 85 90 95 Gly Ala Val Val GlyGly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met 100 105 110 Ser Arg Pro IleIle His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu AsnMet His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Met 130 135 140 Asp GluTyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170175 Thr Glu Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180185 190 Val Thr Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg195 200 205 Ser 7 209 PRT Artificial Sequence Description of ArtificialSequence ChV type prion protein 7 Lys Lys Arg Pro Lys Pro Gly Gly TrpAsn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser Pro Gly GlyAsn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp Gly Gln Pro His GlyGly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly Gly Trp Gly Gln Pro His GlyGly Gly Trp Gly Gln Pro His Gly 50 55 60 Gly Gly Trp Gly Gln Gly Gly GlyThr His Ser Gln Trp Asn Lys Pro 65 70 75 80 Ser Lys Pro Lys Thr Asn MetLys His Met Ala Gly Ala Ala Ala Ala 85 90 95 Gly Ala Val Val Gly Gly LeuGly Gly Tyr Val Leu Gly Ser Ala Met 100 105 110 Ser Arg Pro Ile Ile HisPhe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu Asn Met HisArg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Met 130 135 140 Asp Glu Tyr SerAsn Gln Asn Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160 Thr IleLys Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170 175 ThrGlu Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180 185 190Val Thr Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg 195 200205 Ser 8 765 DNA Mus musculus CDS (1)..(765) 8 atg gcg aac ctt ggc tactgg ctg ctg gcc ctc ttt gtg act atg tgg 48 Met Ala Asn Leu Gly Tyr TrpLeu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 act gat gtc ggc ctc tgcaaa aag cgg cca aag cct gga ggg tgg aac 96 Thr Asp Val Gly Leu Cys LysLys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 acc ggt gga agc cgg tat cccggg cag gga agc cct gga ggc aac cgt 144 Thr Gly Gly Ser Arg Tyr Pro GlyGln Gly Ser Pro Gly Gly Asn Arg 35 40 45 tac cca cct cag ggt ggc acc tggggg cag ccc cac ggt ggt ggc tgg 192 Tyr Pro Pro Gln Gly Gly Thr Trp GlyGln Pro His Gly Gly Gly Trp 50 55 60 gga caa ccc cat ggg ggc agc tgg ggacaa cct cat ggt ggt agt tgg 240 Gly Gln Pro His Gly Gly Ser Trp Gly GlnPro His Gly Gly Ser Trp 65 70 75 80 ggt cag ccc cat ggc ggt gga tgg ggccaa gga ggg ggt acc cat aat 288 Gly Gln Pro His Gly Gly Gly Trp Gly GlnGly Gly Gly Thr His Asn 85 90 95 cag tgg aac aag ccc agc aaa cca aaa accaac ctc aag cat gtg gca 336 Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr AsnLeu Lys His Val Ala 100 105 110 ggg gct gcg gca gct ggg gca gta gtg gggggc ctt ggt ggc tac atg 384 Gly Ala Ala Ala Ala Gly Ala Val Val Gly GlyLeu Gly Gly Tyr Met 115 120 125 ctg ggg agc gcc gtg agc agg ccc atg atccat ttt ggc aac gac tgg 432 Leu Gly Ser Ala Val Ser Arg Pro Met Ile HisPhe Gly Asn Asp Trp 130 135 140 gag gac cgc tac tac cgt gaa aac atg taccgc tac cct aac caa gtg 480 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr ArgTyr Pro Asn Gln Val 145 150 155 160 tac tac agg cca gtg gat cag tac agcaac cag aac aac ttc gtg cac 528 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser AsnGln Asn Asn Phe Val His 165 170 175 gac tgc gtc aat atc acc atc aag cagcac acg gtc acc acc acc acc 576 Asp Cys Val Asn Ile Thr Ile Lys Gln HisThr Val Thr Thr Thr Thr 180 185 190 aag ggg gag aac ttc acc gag acc gatgtg aag atg atg gag cgc gtg 624 Lys Gly Glu Asn Phe Thr Glu Thr Asp ValLys Met Met Glu Arg Val 195 200 205 gtg gag cag atg tgc gtc acc cag taccag aag gag tcc cag gcc tat 672 Val Glu Gln Met Cys Val Thr Gln Tyr GlnLys Glu Ser Gln Ala Tyr 210 215 220 tac gac ggg aga aga tcc agc agc accgtg ctt ttc tcc tcc cct cct 720 Tyr Asp Gly Arg Arg Ser Ser Ser Thr ValLeu Phe Ser Ser Pro Pro 225 230 235 240 gtc atc ctc ctc atc tcc ttc ctcatc ttc ctg atc gtg gga tga 765 Val Ile Leu Leu Ile Ser Phe Leu Ile PheLeu Ile Val Gly 245 250 255 9 254 PRT Mus musculus 9 Met Ala Asn Leu GlyTyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr Asp Val GlyLeu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly SerArg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro GlnGly Gly Thr Trp Gly Gln Pro His Gly Gly Gly Trp 50 55 60 Gly Gln Pro HisGly Gly Ser Trp Gly Gln Pro His Gly Gly Ser Trp 65 70 75 80 Gly Gln ProHis Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 85 90 95 Gln Trp AsnLys Pro Ser Lys Pro Lys Thr Asn Leu Lys His Val Ala 100 105 110 Gly AlaAla Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120 125 LeuGly Ser Ala Val Ser Arg Pro Met Ile His Phe Gly Asn Asp Trp 130 135 140Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val 145 150155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr ThrThr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met GluArg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu SerGln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser Ser Thr Val Leu PheSer Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile Ser Phe Leu Ile PheLeu Ile Val Gly 245 250 10 200 PRT Homo sapiens 10 Lys Lys Arg Pro LysPro Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln GlySer Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp GlyGln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly Gly Trp GlyGln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly 50 55 60 Thr His Ser GlnTrp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys 65 70 75 80 His Met AlaGly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly 85 90 95 Gly Tyr MetLeu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe Gly 100 105 110 Ser AspTyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro 115 120 125 AsnGln Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn 130 135 140Phe Val His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr 145 150155 160 Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met165 170 175 Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg GluSer 180 185 190 Gln Ala Tyr Tyr Gln Arg Gly Ser 195 200

1. A screening method for infectious agents of human or non-human priondiseases in a sample, which employs, as an indication, the deposition ofan aberrant prion protein in the follicular dendritic cell (FDC) of anon-human animal.
 2. The screening method for according to claim 1,wherein the non-human animal is a transgenic animal that expresses ahumanized prion protein gene.
 3. The screening method for according toclaim 1, wherein the non-human animal is a knock-in animal thatexpresses a humanized prion protein gene.
 4. The screening method foraccording to any one of claims 1 to 3, wherein the sample isadministered intraperitoneally, intracerebrally, intravascularly, ororally to the non-human animal.
 5. The screening method for according toany one of claims 1 to 4, wherein the deposition of the aberrant prionprotein in the FDC is detected by a histological detection method,electrophoresis, and/or binding assay.
 6. The screening method foraccording to any one of claims 1 to 5, wherein the deposition of theaberrant prion protein in the FDC is detected 14 to 700 days after thesample administration.
 7. The screening method for according to any oneof claims 1 to 6, wherein the humanized prion protein is prepared bysubstituting a part of the exon 3 of the non-human animal prion proteingene with a part of the exon 3 of the human prion protein.
 8. Thescreening method for according to any one of claims 1 to 6, wherein thehumanized prion protein is prepared by substituting 6 residues on theC-terminal side among human-specific amino acid residues in the humanprion protein with corresponding amino acid residues in the non-humananimal prion protein that is used in the screening.
 9. The screeningmethod for according to any one of claims 1 to 6, wherein the humanizedprion protein comprises an amino acid sequence as shown in SEQ ID NO: 6or
 7. 10. A recombinant humanized prion protein, which is encoded by arecombinant gene in which a part of the exon 3 of the non-human animalprion protein gene is substituted with a part of the exon 3 of the humanprion protein gene.
 11. A recombinant humanized prion protein, wherein 6residues on the C-terminal side among human-specific amino acid residuesin the human prion protein are substituted with corresponding aminoacids in non-human animal prion protein.
 12. A recombinant humanizedprion protein, which comprises an amino acid sequence as shown in SEQ IDNO: 6 or
 7. 13. A gene or a fragment thereof, which encodes the proteinaccording to any one of claims 10 to
 12. 14. A gene or a fragmentthereof, which comprises a nucleotide sequence as described in thefollowing: (a) a nucleotide sequence as shown in SEQ ID NO: 4; (b) anucleotide sequence that is a degenerate sequence of the nucleotidesequence as shown in SEQ ID NO: 4; or (c) a nucleotide sequence that ishybridizable with the sequence according to (a) or (b) under stringentconditions and encodes the protein according to claim
 10. 15. A vector,which comprises the gene or a fragment thereof according to claim 13 or14.
 16. A transgenic animal having the gene or a fragment thereofaccording to claim 13 or 14 introduced therein.
 17. A knock-in animalhaving the gene or a fragment thereof according to claim 13 or 14introduced therein.
 18. A transgenic animal, which expresses a humanizedprion protein in the FDC.
 19. A knock-in animal, which expresses ahumanized prion protein in the FDC.
 20. A transgenic animal, whichexpresses the protein according to any one of claims 10 to 12 in itsbrain and/or FDC.
 21. A knock-in animal, which expresses the proteinaccording to any one of claims 10 to 12 in its brain and/or FDC.
 22. Amethod for producing a transgenic animal, which expresses the proteinaccording to any one of claims 10 to
 12. 23. A method for producing aknock-in animal, which expresses the protein according to any one ofclaims 10 to
 12. 24. The method for producing a knock-in animalaccording to claim 23 comprising the following steps of: (a)constructing a vector containing a non-human prion protein gene or afragment thereof; (b) substituting a part of the exon 3 of the non-humanprion protein gene with a part of the exon 3 of a human prion protein;(c) inserting a loxp-surrounded antibiotic-resistant gene into the3′-non-translation region; (d) introducing the resulting modified vectorinto the non-human ES cell; (e) producing a chimera animal from a clonehaving homologous recombination; and (f) removing theantibiotic-resistant gene by introducing a Cre enzyme-expressing plasmidinto a fertilized egg of the F1 animal.
 25. A screening method for apreventive and/or therapeutic agent for human or non-human priondiseases, which utilizes the transgenic animal according to claim 18 or20 or the knock-in animal according to claim 19 or 21.